Characterization of a Novel α-Conotoxin from Conus textile That Selectively Targets α6/α3β2β3 Nicotinic Acetylcholine Receptors*

Background: Cone snails are a rich source of α-conotoxins that target nicotinic acetylcholine receptors (nAChR). Results: A new α-conotoxin TxIB potently blocked α6/α3β2β3 nAChRs with high selectivity. Conclusion: TxIB is an effective inhibitor of α6/α3β2β3 nAChRs. Its structure was determined by NMR. Significance: TxIB is a new, uniquely selective ligand for querying the structure and function of nAChRs and designing therapeutic drugs. α6β2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the β4 subunit to form α6β4 nAChRs that are difficult to pharmacologically distinguish from α6β2, α3β4, and α3β2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3β2β3 nAChR with an IC50 of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 μm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6β2 nAChRs.

␣6␤2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the ␣6 subunit can also associate with the ␤4 subunit to form ␣6␤4 nAChRs that are difficult to pharmacologically distinguish from ␣6␤2, ␣3␤4, and ␣3␤2 subtypes. The current study characterized a novel 16-amino acid ␣-conotoxin (␣-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. ␣-CTx TxIB blocked ␣6/␣3␤2␤3 nAChR with an IC 50 of 28 nM. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 M. The three-dimensional solution structure of ␣-CTx TxIB was determined using NMR spectroscopy. ␣-CTx TxIB represents a uniquely selective ligand for probing the structure and function of ␣6␤2 nAChRs.
Nicotinic acetylcholine receptors (nAChRs) 2 are ligand gated ion channels distributed throughout the nervous system.
In addition, they are increasingly recognized as fundamental macromolecules in non-neuronal systems (1). nAChRs are assembled from ␣ (␣1-␣10) and/or ␤ (␤1-␤4) subunits to form a variety of subtypes that have distinct pharmacological properties and physiological functions. Expression of the ␣6 subunit has previously been thought to be primarily localized to catecholaminergic nuclei of the central nervous system. However, recent evidence indicates that the ␣6 subunit is abundantly expressed in visual pathways and is also present in peripheral tissues (2)(3)(4).
As the recognized anatomical sites of expression of ␣6 receptors has grown, so too has the complexity of recognized subtypes. Work to date has largely focused on ␣6␤2* nAChRs that modulate the release of dopamine. These receptors are believed to be important in mediating tobacco addiction and are also implicated in the pathophysiology of Parkinson disease (5). Furthermore, ␣6* nAChRs containing the ␤4 rather than or in addition to the ␤2 subunit have recently been reported in a variety of tissues including retina (6 -7), hippocampus (8), dosrsal root ganglion (3), and adrenal chromaffin cells. However, there is a paucity of ligands that can effectively discriminate between ␣6␤2 and ␣6␤4 nAChRs.
Toxin-producing organisms have served as a natural source of molecules that target nAChRs. Plants and animals use these toxins defensively to discourage consumption by predators or offensively to immobilize prey. Marine mollusks of the genus Conus fall into the latter category and produce an arsenal of neuroactive peptides, many of which are targeted to nAChRs. ␣-Conotoxins are small cysteine-rich peptide toxins that contain two or more disulfide bonds. ␣-Conotoxin MII from Conus magus is a signature antagonist of ␣6␤2 nAChRs (9). However, this ligand also binds to ␣3␤2 and ␣6␤4 nAChRs, highlighting the need for development of further ligands (10 -14). Here, we report the discovery of a novel toxin from C. textile that binds with high affinity to ␣6/␣3␤2␤3 nAChRs but not ␣3␤2 or ␣6␤4 nAChRs. The new toxin was synthesized and we report its pharmacological profile and three-dimensional solution structure as determined by NMR.
Identification and Sequencing of a Genomic DNA Clone Encoding ␣-Conotoxin TxIB-Genomic DNA from the C. textile venom gland was isolated using a marine animal DNA Isolation Kit (Tiangen Biochemistry Ltd., Beijing, China). The procedure followed the manufacturer's suggested protocol for marine invertebrates as described previously (17). Briefly, frozen tissue (less than 30 mg) was placed in 200 l of lysis buffer with 40 l of RNase A (10 mg/ml), vortexed for 15 s, and digested for 1.5 h with 20 l of 20 mg/ml of proteinase K solution at 56°C. The remainder of the procedure followed the kit protocol. The resulting genomic DNA was used as a template for PCR using oligonucleotides primers, corresponding to the 3Ј-end of the intron preceding the toxin region of ␣-conotoxin prepropeptides (Primer 1) and the 3Ј-UTR (untranslated region) sequence of ␣-conotoxin prepropeptides (Primer 2). The sequence of Primer 1 was 5Ј-GTGGTTCTGGGTC-CAGCA-3Ј. The sequence of Primer 2 was 5Ј-GTCGTG-GTTCAGAGGGTC-3Ј. The PCR amplification was performed as described previously (17). PCR products were analyzed by electrophoresis on 1.5% agarose gel. The PCR products were purified by AxyPrep-PCR & Gel Clean up (Axygen Bio. Ltd., Hangzhou, China), which were inserted into the pGEM-T Easy vector via TA cloning (TaKaRa). Transformed colonies were screened with white-blue identification for sequence analysis. Plasmids of positive transformed colonies with conopeptide precursor DNA inserts were purified with a UNlQ-10 EZ Spin Column Plasmid Mini-Preps Kit (Yili Bioscience Ltd., Shanghai, China). Purified plasmids were sequenced by Sangon Ltd.
Chemical Synthesis-The peptide was assembled on an amide resin by solid-phase methodology on an ABI 433A peptide synthesizer using FastMoc (N-(9-fluorenyl) methoxycarbonyl) chemistry and standard side chain protection, except for cysteine residues. Cysteine residues were protected in pairs with either S-trityl on Cys 2 and Cys 8 , or S-acetamidomethyl on Cys 3 and Cys 16 . The peptides were removed from a solid support by treatment with reagent K (trifluoroacetic acid/water/ ethanedithiol/phenol/thioanisole; 90:5:2.5:7.5:5, v/v). The released peptide was precipitated and washed several times with cold ether. A two-step oxidation protocol was used to fold the peptides selectively as described previously (18). Briefly, the disulfide bond between Cys 2 and Cys 8 was oxidized by adding the peptide into an equal volume of 20 mM potassium ferricyanide, 0.1 M Tris, pH 7.5. The solution was allowed to react for 45 min, and the monocyclic peptide was purified by reverse-phase HPLC. Simultaneous removal of the S-acetamidomethyl groups and oxidation of the disulfide bond between Cys 3 and Cys 16 were carried out by iodine oxidation as follows: the monocyclic peptide in HPLC eluent was dripped into an equal volume of iodine (10 mM) in H 2 O:trifluoroacetic acid:acetonitrile (73:3:24 by volume) and allowed to react for 10 min. The reaction was terminated by the addition of ascorbic acid and the bicyclic peptide was purified by HPLC on a reversed phase C18 Vydac column using a linear gradient of ACN: 0 -40 min 2-42% solvent B (solvent B is 0.092% trifluoroacetic acid in 60% ACN; solvent A is 0.1% trifluoroacetic acid in H 2 O). Matrixassisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was utilized to confirm the identity of the products.
cRNA Preparation and Injection-Capped cRNA for the various subunits were made using the mMessage mMachine in vitro transcription kit following linearization of the plasmid. The cRNA was purified using the Qiagen RNeasy kit. The concentration of cRNA was determined by absorbance at 260 nm. cRNAs of the various subunits were combined to give 200 -500 ng/l of each subunit cRNA. Fifty nl of this mixture was injected into each Xenopus oocyte as described previously, and incubated at 17°C. Oocytes were injected within 1 day of harvesting and recordings were made 2-4 days post-injection.
Voltage-clamp Recording-Oocytes were voltage-clamped and exposed to ACh and peptide as described previously (10). Briefly, the oocyte chamber consisting of a cylindrical well (ϳ30 l in volume) was gravity perfused at a rate of ϳ2 ml/min with ND-96 buffer (96.0 mM NaCl, 2.0 mM KCl, 1.8 mM CaCl 2 , 1.0 mM MgCl 2 , 5 mM HEPES, pH 7.1-7.5) containing 1 M atropine and 0.1 mg/ml of BSA. In the case of the ␣9␣10, ␣7, and mouse muscle ␣1␤1␦⑀ subtypes, the ND96 contained no atropine during recording. The membrane potential of the oocytes was clamped at Ϫ70 mV. The oocyte was subjected once a minute to a 1-s pulse of ACh. ACh concentration was 10 M for ␣9␣10 and ␣1␤1␦⑀ subtypes, 200 M for ␣7, and 100 M for all other subtypes. Once a stable baseline was achieved, either ND-96 alone or ND-96 containing varying concentrations of the ␣-conotoxins were pre-applied for 5 min prior to the addition of the agonist. All recordings were done at room temperature (ϳ22°C).
Data Analysis-The average of five control responses just preceding a test response was used to normalize the test response to obtain "% response." Each data point of a doseresponse curve represents the average Ϯ S.E. of at least three oocytes. The dose-response data were fit to the equation, % response ϭ 100/[1 ϩ ([toxin]/IC50) ϫ n H ], where n H is the Hill coefficient, by nonlinear regression analysis using GraphPad Prism (GraphPad Software, San Diego, CA).
NMR Spectroscopy-TxIB (3.0 mg) was dissolved in 550 l of 10% D 2 O, 90% H 2 O (ϳpH 3.5) and spectra were recorded on a Bruker Avance 600 MHz NMR spectrometer. One-dimensional and two-dimensional NMR experiments including TOCSY, NOESY, and double-quantum filtered-correlated spectroscopy spectra were recorded at 280 K. Chemical shifts were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate at 0 ppm. The TOCSY and NOESY spectra were run with mixing times of 80 and 200 ms, respectively. Two-dimensional spectra were generally collected over 4096 data points in the f2 dimension and 512 increments in the f1 dimension over a spectral width of 12 ppm. Processed spectra were analyzed and assigned using the program CCPNMR with the sequential assignment protocol (19).
Structure Calculations-Distance restraints were derived from the NOESY spectrum using peak volumes. Dihedral angle restraints were determined from 3 J HN-H␣ coupling constants obtained from one-dimensional 1 H NMR spectrum. The angles were restrained to Ϫ60 Ϯ 30 o for 3 J HN-H␣ Ͻ 5.8 Hz and Ϫ120 Ϯ 30 o for 3 J HN-H␣ Ͼ 8.0 Hz. Preliminary structures were generated using CYANA (20) and the 50 final structures were generated within CNS (21) using protocols from the RECO-ORD database (22). The final structures were analyzed for stereochemical quality using MOLPROBITY (23) and the 20 of with lowest energy and best quality were chosen to represent the solution structure of ␣-CTx TxIB.

RESULTS
Gene Cloning of the Novel ␣-CTx TxIB-In the venom of most analyzed cone snail species there are multiple ␣-conotoxins targeted to nAChRs. Although the mature ␣-conotoxin sequences diverge considerably, the organization of ␣-conotoxin genes reveals substantial conservation across species. The intron immediately preceding the toxin sequence, the signal sequence within the exon, and the 3Ј-untranslated region show a high degree of conservation. Proteolytic processing of the precursor protein leads to the formation of the mature ␣-conotoxin. The coding region of the signal sequence and most of the propeptide region is separated from the toxin-coding region and 3Ј-untranslated region by a single intron. Primers were designed to this conserved intron sequence and used in conjunction with primers to the conserved 3Ј-untranslated region to PCR amplify the toxin-coding portion of the ␣-conotoxin gene. PCR amplification of genomic DNA from C. textile yielded the ␣-CTx gene product, which was cloned and sequenced. The DNA fragment was 170 bp in length and encoded an ␣-conotoxin precursor representing the ␣-conotoxin TxIB gene (tx1b). The precursor was a 47-amino acid protein (Fig. 1). This sequence displayed the characteristic structural organization of conotoxins, including a propeptide sequence at the N terminus, followed by the mature toxin region and a closing 3Ј-UTR (24). The predicted mature peptides exhibited the characteristic cysteine pattern of ␣-CTxs (CC-C-C). The EMBL accession number of the ␣-CTx TxIB precursor gene (tx1b) from C. textile is HE995411. The ␣-CTx precursor contained an N-terminal propeptide having a length of 27-amino acids and was marked by a predominance of basic residues. It was separated from the proregion by the proteolytic site-XR, which would yield a peptide with a length of 20 amino acids, and further be truncated to a length of 16 (␣-CTx TxIB) or 17 (␣-CTx TxIB(G)) amino acids by removal of a C-terminal glycine during C-terminal amidation (Fig. 1). The first or second glycine following the C-terminal cysteine in the mature toxin is presumed to be processed to a C-terminal amide for TxIB or TxIB(G), respectively. The deduced mature toxin sequence of TxIB is GCCSDPPCRNKHPDLC# (# ϭ C-terminal carboxamide). TxIB(G) is an analog of ␣-CTx TxIB with an extra glycine residue.
Because members of each conopeptide gene family share a highly conserved signal and propeptide sequence and a conserved arrangement of the cysteines in the sequence of the mature toxin, the new conopeptides TxIB and TxIB(G) identified in this work can be designated as members of the ␣-conotoxin family. Some selected ␣-conotoxin precursors and mature toxins from various Conus species are shown for comparison in Fig. 2 and Table 1. Note the highest sequence homol-FIGURE 1. ␣-Conotoxin TxIB prepropeptide and encoded toxin are shown. A putative proteolytic processing site 1 (R) and amidated processing sites 2 and 3 are indicated by arrows. The pro-region is italics. The mature toxin region is underlined. The first and second glycine following the C-terminal cysteine in the mature toxin is presumed to be processed to a C-terminal amide for ␣-CTx TxIB and ␣-CTx TxIB(G), respectively. TxIB(G) is an analog of TxIB with an extra G (TxIB(G)). The cysteine residues are indicated in bold. The stop codon is indicated by the ᭜. The deduced mature toxin sequence of TxIB is GCCSDPPCRNKHPDLC# (# ϭ C-terminal carboxamide). ␣-CTx TxIB Selectively Blocks ␣6/␣3␤2␤3 nAChRs ogy in the signal and 3Ј-UTR, higher sequence homology in the propeptide region and, in contrast, divergence in the mature toxin region particularly in the C-terminal portion of the peptides. Because the intron is located within the pro-region immediately preceding the toxin sequence, the ␣-conotoxin cloned from genomic DNA lacks the signal sequence from the toxin-containing fragment (Table 1).
Chemical Synthesis and Oxidative Folding-Fmoc (N-(9fluorenyl)methoxycarbonyl) chemistry was used to synthesize the linear peptides. With four cysteine residues there are three possible disulfide bond connectivities. Previously characterized ␣-conotoxins purified from venom typically have a disulfide bond connectivity linking cysteine I to III and II to IV, which is referred to as the "globular" form to distinguish it from the alternative "ribbon" or "beads" connectivities (25)(26). We therefore synthesized both ␣-CTx TxIB and TxIB(G) in the globular form using a regioselective oxidation approach (Fig. 2). We protected the cysteine side chains with two orthogonal protecting groups that could be removed selectively under different conditions, allowing the formation of one disulfide bridge at a time. For this purpose, Cys 2 and Cys 8 were introduced as the acid labile S-trityl protected amino acids, whereas Cys 3 and Cys 16 were S-acetamidomethyl cysteine. The acid-labile groups were removed simultaneously with cleavage from the resin; ferricyanide was used to oxidize the first disulfide bond. Reversephase HPLC was used to purify the monocyclic peptide; subsequently, the acid-stable acetamidomethyl groups were removed from the second and fourth cysteines by iodine oxidation, which simultaneously formed the second disulfide bond. The two fully oxidized peptides were purified again by HPLC. Laser desorption mass spectra of synthetic ␣-CTx TxIB and TxIB(G) were consistent with the sequences. TxIB monoisotopic MS was: calculated 1739.70 and observed 1739.6. TxIB(G) monoisotopic MS was: calculated 1796.72 and observed 1796.6. Synthetic peptides with Cys 2 -Cys 8 ; Cys 3 -Cys 16 disulfide bond arrangement were used in all subsequently described studies.
NMR Spectroscopy-For NMR analysis, one-and two-dimensional spectra were recorded at 280 K and assigned using well established techniques (19). The fingerprint region in the NOESY spectrum of the peptide shows a complete cycle of H␣-NH iϩ1 sequential connectivities with the exception of the three proline residues (Pro 6 , Pro 7 , and Pro 13 ). However, as expected, NOEs were observed from the H␣-H␦ iϩ1 protons of the Pro 6 and Pro 13 residues and their preceding residues. Residue Pro 7 was assigned based on a NOE peak between the H␤s of Pro 6 and H␦ of Pro 7 . Chemical shifts for all residues are given in Table 3.
Structure Determination of ␣-CTx TxIB-A set of 50 structures was calculated using a simulated annealing protocol in the CNS based on 133 distance restraints derived from NOESY cross-peaks, including 46 intraresidue, 49 sequential, 32 medium range (1Ͻ ͉iOj͉ Ͻ 5) and 6 long range (͉iOj͉ Ն 5) NOEs, and 7 dihedral restraints based on coupling constants. The calculated structures were consistent with the experimental data, with no NOE violations greater than 0.2 Å and no dihedral violations greater than 3.0 o . The 20 lowest energy structures (Fig. 5) were selected based on the highest overall MOLPROBITY scores and had a global root mean square deviation of 0.70 Ϯ 0.21 Å across backbone residues 2-16. A summary of the energy and MOLPROBITY statistics is given in Table 4. As illustrated in Fig. 5B, the secondary structure of TxIB comprises an ␣-helix between residues 6 and 9, as is very common in ␣-conotoxins. The PDB accession number of the ␣-CTx TxIB structure is 2LZ5.

DISCUSSION
C. textile hunts snails and its prey includes numerous species of prosobranch gastropods. When faced with starvation, C. textile will practice cannibalism. The venom is toxic to vertebrates and is reported to have caused human fatalities (28). The Conus genus as a whole hunts a wide variety of prey from five different phyla. These diverse feeding habits are mirrored by the diversity and complexity of the venom components of individual species. In contrast, the coding region of the signal sequence and the propeptide region of the ␣-CTxs are separated from the toxin-coding region and 3Ј-untranslated region by a single intron. In this study, by using a conserved intron sequence found within the genes that encode ␣-conotoxin precursors, a PCR based technique was used to identify a novel peptide from the snail hunting species C. textile. We note that native ␣-CTx TxIB has not been isolated from venom. Therefore, both ␣-CTx TxIB and ␣-CTx TxIB(G) peptides were chemically synthesized After control responses to ACh, the oocyte was exposed to 1 M ␣-CTx TxIB for 5 min. Toxin was then washed out and the response to ACh was again measured (arrow). B, concentration response of ␣6/␣3␤2␤3 nAChRs exposed to ␣-CTx TxIB and ␣-CTx TxIB(G). Values shown in the graph are mean Ϯ S.E. from 3-10 separate oocytes.
Ͼ10,000 b a Numbers in parentheses are 95% confidence intervals. b Less than 50% block at 10 Ϫ5 M. All receptors are rat except for ␣1␤1␦⑀, which is mouse.
␣-CTx TxIB Selectively Blocks ␣6/␣3␤2␤3 nAChRs using the disulfide bond configuration of previously characterized ␣-conotoxins, that is, with a I-III, II-IV disulfide connectivity (29). The residues between Cys-II and Cys-III and Cys-III and Cys-IV of ␣-conotoxins are commonly referred to as loops 1 and 2, respectively. The number of residues in each of these loops is used to further classify the ␣-conotoxins. For example, TxIB is classified as a 4/7 ␣-conotoxin, whereas the muscle nAChR selective ␣-CTx MI has a 3/5 spacing (Table 1). Although TxIB has a highly conserved Ser-Xaa-Pro motif in loop 1 that is crucial for potent nAChR interaction, the amino acids "RNKH" in loop 2 are distinct, suggesting that amino acids in the C-terminal half of ␣-CTx TxIB might be responsible for its selectivity.
␣-CTx TxIB potently blocked ␣6/␣3␤2␤3 nAChRS but had little or no activity at other tested subtypes. The ability of ␣-CTx TxIB to discriminate between ␣6/␣3␤2␤3 and the other nAChR receptors is unique. No small molecules have this selectivity profile. Previously described ␣-conotoxins that potently block ␣6/␣3␤2␤3 nAChRs also block ␣6/␣3␤4 nAChRs, ␣3␤2 nAChRs, and/or other nAChRs subtypes. For instance, another 4/7 conotoxin, ␣-CTx MII from the fish-eating C. magus, potently blocks ␣6/␣3␤2␤3 and ␣3␤2 nAChRs, with weaker activity on ␣6/␣3␤4 nAChRs. A second generation analog of ␣-CTx MII, ␣-CTx MII(H9A,L15A) loses activity at the ␣3␤2 nAChR subtype, but retains activity on the ␣6␤4 subtype (14). Another 4/7 ␣-CTx, PIA, is an 18-amino acid peptide from the fish eating snail, Cyclamen purpurascens. PIA is most potent on   ␣-CTx TxIB Selectively Blocks ␣6/␣3␤2␤3 nAChRs ␣6/␣3␤2␤3 nAChR but also potently blocks ␣6/␣3␤4 nAChRs (18). ␣-CTx BuIA is a 13-amino acid 4/4 peptide from the fish eating snail, Conus bullatus. Although it potently blocks ␣6/␣3␤2␤3 nAChRs, it is also active against a broad spectrum of nAChR subtypes, including ␣6/␣3␤4, ␣3␤2, and ␣3␤4 (30). The kinetics of toxin unblock for the latter three toxins are slow. In contrast, the TxIB block is rapidly reversed after toxin washout, and in this way is more similar to the peptide ␣-CTx GIC that potently blocks both ␣6/␣3␤2␤3 and ␣3␤2 nAChRs (31). NMR and structural analyses showed that TxIB has a similar fold to other ␣-CTxs. As illustrated in Fig. 6, even though they have the same peptide fold, conotoxins MII, PIA, BuIA, GIC, and TxIB have different surface features in terms of the charged, polar, and hydrophobic side chains of the residues. It is clear that all of the peptides have a hydrophobic patch in loop 1. However, a discriminating feature of TxIB is that the hydrophobic patch is smaller than in MII, PIA, or BuIA, suggesting the possibility that this feature might be responsible for the higher selectivity of TxIB. However, the hydrophobic patch in GIC is also rather small, suggesting that this cannot be the only factor. A main difference between GIC and TxIB is the presence of two positive charges flanking the hydrophobic patch in TxIB. In the absence of a detailed molecular model defining the orientation of the toxins bound to the receptor it is difficult to be definitive about the reasons for the trends in specificity, but it appears likely the combination of smaller hydrophobic patch with flanking positively charged residues helps to enhance the selectivity of TxIB. Note that the presence of positive charge alone is not sufficient to confer selectivity as conotoxin PIA, which has a positive charge at R1, is nonspecific. In future studies this hypothesis could be further tested by the synthesis of derivatives of BuIA or PIA where mutations are made in the hydrophobic patches and in flanking residues.
The nAChR ␣6 subunit is not widely expressed in the brain, but it is abundant in midbrain dopaminergic regions associated with pleasure, reward, and mood control (32)(33)(34)(35), suggesting that ␣6*-nAChRs might play critical roles in nicotinic reward and in the regulation of mood by nicotine (36). Modulation of striatal dopamine neurotransmission plays a key role in the reinforcing effects of addictive drugs (35,37). Striatal dopamine  ␣-CTx TxIB Selectively Blocks ␣6/␣3␤2␤3 nAChRs axon terminals express ␣6␤2 and ␣4(non-␣6)␤2* nAChRs. Pharmacological block of the ␣6␤2 nAChR function is associated with decreased self-administration of nicotine as well as ethanol in rat models of substance abuse (38 -40). In addition, genetic deletion of the nAChR ␣6 subunit abolishes nicotine self-administration and lentiviral re-expression of the ␣6 subunit leads to restoration of nicotine self-administration (35). Thus, ␣6␤2* nAChR represents a potentially attractive drug target to treat substance dependence. Although TxIB itself is unlikely to cross the blood-brain barrier in significant quantity, the discovery of ␣-CTx TxIB represents proof-of-concept that it is possible to develop compounds that selectively block ␣6␤2 nAChRs. ␣-CTx TxIB might therefore represent an initial scaffold from which to design therapeutic drugs.
In conclusion, this study describes a novel ␣-conotoxin TxIB that is a subtype-specific blocker of the ␣6/␣3␤2␤3 nAChR. The unique selectivity of this peptide will allow probing of nAChR function in tissues where both the ␣6* and other nAChR subtypes occur. In addition, structural insights derived from this ligand might facilitate the development of novel therapeutics for diseases involving ␣6* nAChRs, including addiction.